The packaging of DNA into chromatin is now widely recognized as a key step in gene regulation in eukaryotic cells. However, relatively little is known about the structure of transcriptionally active vs. repressed chromatin states, or the mechanisms by which these two states might be interchanged. The long term goal of this research is to understand how chromatin is organized into functional domains. Experiments in this proposal will address this question through an analysis of a model regulator in yeast, the Tup1p repressor. Experiments completed in the last funding period have led us to the hypothesis that Tup1p organizes chromatin to repress transcription. We have shown that Tup1p interacts specifically with the amino terminal tail domains of histones H3 and H4 in vitro, and that mutations in these tails domains synergistically compromise repression in vivo. Preliminary data indicate that post-translational acetylation of H3 and H4 inhibits Tup1p binding to these histones, suggesting that this modification may modulate Tup1p functions. Consistent with this, genes subject to Tup1p repression are associated with less acetylated forms of these histones in vivo under conditions of repression than under conditions of activation. Moreover, mutations in specific histone deacetylase activities that cause hyperacetylation in vivo compromise Tup1p repression of multiple target genes. Recent data also indicate that phosphorylation of H3 may affect Tup1p functions. Mutation of a conserved phosphorylation site in the amino terminal region of H3 (serine 10) severely limits repression. Together, these data suggest a mechanism by which Tup1p-mediated repression might be modulated, and more globally, indicate that one important role of histone modifications may be to regulate the association of regulatory factors with chromatin. Such affects are understudied at present, but may be centrally important to the organization of functional chromatin domains. Experiments in this proposal will further test our hypothesis with two specific aims: 1) to dissect the molecular nature and functional consequences of Tup1p interactions with chromatin and 2) to determine the effects of specific histone modifications on Tup1p functions, as well as how different modification states are established at Tup1p regulated promoters in vivo. Understanding how regulatory proteins like Tup1p influence chromatin structure, and how such functions are themselves modulated, is crucial to understanding how gene expression is programmed in normal cells, and how changes in these functions lead to abnormal transcription patterns associated with disease states.